1. Field of the Invention
The present invention relates to an improvement in a so-called integrated inspection apparatus, featuring the integration of a scanning electron microscope (SEM) and light microscope optics. The improvement in particular relates to extension in functionality of such integrated systems, amongst others towards a new and simplified method of operating such systems.
In this respect, it is noted that information obtained from images with light microscopy and electron microscopy is to a large extent complementary. With a light microscope different objects can be seen and inspected in a specimen in different colors, which allows for identification of part or whole of the composition of this specimen. Instead of directly observing color from a constituent of the specimen, very often specific color markers are attached, such as fluorophores or autofluorescent proteins, to a specific non-colored constituent for identification.
With an electron microscope, all constituents of a specimen can be imaged at very small detail (high resolution), much smaller than with a light microscope, but the ability to identify constituents based on color is absent. In correlative light-electron microscopy, therefore, users try to obtain images from the same area of a specimen, the so called Region of Interest or ROI for short, with both the light and the electron microscope. A very accurate and quick way of doing this, is by using an integrated microscope wherein both types of microscope or parts thereof are to a more or less integrated extend contained in a single apparatus.
2. Description of the Related Art
Such an integrated system is known in the art, e.g. from the short technical note “Specimen stage incorporating light microscopical optics for a Cambridge S180 scanning electron microscope” by Wouters et al in J. of Microscopy Vol. 145, Feb. 1987, pp237-240. Recent improvements of that principle are provided by Applicant in patent publication WO2012008836 and is in line with the present invention generally described as an inspection apparatus provided with an optical microscope and an ion- or electron microscope, equipped with a source for emitting a primary beam of radiation to a sample in a sample holder. The apparatus comprises a detector for detection of secondary radiation backscattered from the sample and induced by the primary beam. The optical microscope is equipped with a light collecting and recording device such as a CCD camera or other light recording device, to receive in use luminescence light emitted by the sample.
In correlative microscopy, users aim to image the same area of a sample with both the light and the electron microscope. The problem in this practice is that both images have different magnification, possibly both in x and y, and rotation orientation. Also, the images have different contrast, which means that some features that are visible in one image cannot be seen in the other, and vice versa. Known methods to overcome this problem are to put the samples on a microscope slide, or support grid, that has markers which can be recognized in both images. Another method is to inject markers into the sample. See for example, “The use of markers for Correlative Microscopy” Brown & Verkade, Protoplasma, 244, pp. 91-94, 2010
One of the essential points of correlative microscopy, i.e. the process of inspecting the same sample with two different investigative methods, is overlaying datasets of the two methods as precisely and accurate possible. In the case of correlative light-electron microscopy, this means an x-, y-, and occasional z-, overlay between an optical image and an electron image. The process of achieving this overlay is non-trivial and may be cumbersome.